Jenway 6715 uv vis spectrophotometer

Total phenolics were determined based on [10 ]. Triplicate 75 mg samples of each biological replicate were vortexed with 1.5 mL acidified methanol and then mixed at 850 rpm on an Eppendorf Thermomixer (Eppendorf Ltd., Stevenage, UK) for 2 h at 23 °C. After centrifugation (Eppendorf Ltd., Stevenage, UK) at 5000× g for 10 min, 1 mL of supernatant was removed into a fresh Eppendorf tube and 200 µL aliquots mixed with 1.5 mL of ×10 diluted Folin–Ciocalteau reagent (Sigma-Aldrich, St. Louis, MO, USA) and left to stand for 5 min. 1.5 mL of 6% (w/v) aqueous sodium carbonate solution was added, mixed and stood at room temperature for 90 min. The absorbance at 725 nm was then measured (Jenway 6715 UV/Vis spectrophotometer, Cole-Parmer, St Neots, UK), and the concentration of phenolics was calculated using ferulic acid as a standard (Sigma-Aldrich) and a standard curve from 20, 40, 100, 150 and 200 µg/mL with three technical replicates of each point.

The EECE were used to determine the extracellular laccase activity by spectrophotometry, monitoring the change in absorbance due to the oxidation of 2,6-dimethoxyphenol (2,6-DMP) using the JENWAY 6715 UV/Vis spectrophotometer (Cole Parmer, Vernon Hills, IL, USA). The standard reaction mixture (1 mL) contained 2,6-DMP (2 mM) in K₂HPO₄ (0.1 M, pH 6.5) and 40 µL of EECE. The reaction was monitored at 468 nm for 4 min at 39 °C [40 (link),41 ]. The enzymatic activity was expressed in International Units (IU), where 1 IU was defined as the amount of enzyme that catalyzes the transformation of 1 µmol of 2,6-DMP into product per minute. The total protein concentration in the EECE was determined following Lowry et al. [42 (link)]. Furthermore, the EECE was used to determine the laccase activity by zymography in semi-denaturing SDS–PAGE in 11% acrylamide. The electrophoresis conditions were 150 V for 1–1.25 h. The zymogram was revealed using 2,6-DMP (2 mM) as substrate in K₂HPO₄ (0.1 M, pH 6.5). The semi-denaturing and denaturing SDS–PAGE gels were stained with colloidal Coomassie brilliant blue (R-250) (Sigma–Aldrich, Waltham, MA, USA).